Chromosomal integrating cassette allowing inducible gene expression for production of compounds via fermentation

Abstract

The present invention provides a new system, resp. method, of genetic modification, called ACIT (Alphaproteobacteria chromosomally inserting transcription-control cassette). The invention, however, is not limited to use in Alphaproteobacteria. The modification can be implemented within the bacterial chromosome and therefore permanently implemented within the microorganism.

1 .- 15 . (canceled) 
     
     
         16 . A transcription control cassette composed for the expression of at least one gene of interest, characterized in that the transcription control cassette comprises the following parts i) through vi),
 i) a first DNA sequence comprising a promoter DNA sequence, being flanked by a first Lac operator DNA sequence and a second Lac operator DNA sequence;   ii) a second DNA sequence encoding for a repressor to bind the Lac operators of i);   iii) a third DNA sequence comprising a second nucleotide promotor sequence to drive expression of ii), wherein the second nucleotide promotor is the same nucleotide promotor as is comprised by part i) or a different one;   iv) a fourth DNA sequence comprising a transcription terminator sequence which is terminating the transcription of part vi;   v) a fifth DNA sequence comprising a sequence for a selection marker;   vi) a sixth DNA sequence comprising at least one fragment from the at least one gene of interest of suitable length which is ensuring the homologous recombination with the chromosomal sequence of the cell into which the transcription control cassette is to be transformed, wherein the fragment from the at least one gene of interest is comprising the first nucleotides of the respective gene of interest;   wherein the DNA sequences of any two or more of parts i) through vi)
 are connected directly to each other within the complete sequence of the transcription control cassette 
   or
 are separated from each other within the complete sequence of the transcription control cassette by DNA sequences of DNA restriction cleavage sites. 
   
     
     
         17 . A transcription control cassette according to  claim 16 , characterized in that the first DNA sequence comprising a promoter DNA sequence according to part i) has at least 50% nucleotide identity with a promotor DNA sequence selected from the list comprising the promotor DNA sequences SEQ ID no. 4, SEQ ID no. 21, SEQ ID no. 22, SEQ ID no. 23, SEQ ID no. 24, SEQ ID no. 25, SEQ ID no. 26, SEQ ID no. 27, SEQ ID no. 28, SEQ ID no. 29, SEQ ID no. 30. 
     
     
         18 . A transcription control cassette according to  claim 16 , wherein the second nucleotide promotor sequence according to part iii) is a DNA sequence having at least 50% nucleotide identity with SEQ ID no. 10 or a DNA sequence having at least 50% nucleotide identity with SEQ ID no. 11. 
     
     
         19 . A transcription control cassette according to  claim 16 , wherein the fourth nucleic acid according to part iv) is a DNA sequence having at least 50% nucleotide identity with SEQ ID no. 9
 and/or   the sequence according to part v) is a DNA sequence having at least 50% nucleotide identity with SEQ ID no. 7.   
     
     
         20 . A transcription control cassette according to  claim 16 , wherein the fragment from the at least one gene of interest according to part vi) is a DNA sequence having at least 50% nucleotide identity with SEQ ID no. 12, SEQ ID no. 12 being a fragment from the gene sinR, coding for the AHL synthase, or having at least 50% nucleotide identity with SEQ ID no. 13, SEQ ID no. 13 being a fragment from the gene expR, coding for an AHL receptor protein, or having at least 50% nucleotide identity with SEQ ID no. 14, SEQ ID no. 14 being a fragment from the gene phaC, coding for the synthase for the production of polyhydroxybutyrate, or having at least 50% nucleotide identity with SEQ ID no. 15, SEQ ID no. 15 being a fragment from the gene mraZ, being the first gene in the dcw gene cluster, the dcw gene cluster being relevant for controlling cell division. 
     
     
         21 . A transcription control cassette according to  claim 16 , characterized in that it comprises a DNA sequence having at least 50% nucleotide identity with SEQ ID no. 5, SEQ ID no. 5 comprising SEQ ID no. 14. 
     
     
         22 . A transcription control cassette according to  claim 16 , characterized in that the activity of the promoter of part i) is inducible by the presence of a compound of initialisation. 
     
     
         23 . A transcription control cassette according to  claim 22 , wherein the compound of initialisation for the transcription control cassette is selected from the list comprising IPTG, allolactose, and galactose thioglycosides. 
     
     
         24 . A vector comprising the transcription control cassette according to  claim 16 . 
     
     
         25 . Usage of the transcription control cassette according to  claim 16  for the homologous or heterologous expression of at least one gene of interest in cells by transforming the transcription control cassette into the cells and cultivating the cells. 
     
     
         26 . Usage according to  claim 25 , characterized in that the at least one gene of interest encodes an enzyme for the production of a fine chemical. 
     
     
         27 . A recombinant bacterial cell comprising the transcription control cassette of  claim 16  and the at least one gene of interest, wherein the at least one gene of interest is operatively linked to the transcription control cassette and the transcription control cassette and the at least one gene of interest are located within the bacterial chromosome. 
     
     
         28 . The bacterial cell according to  claim 27 , characterized in that it is a cell of a gram-negative bacteria. 
     
     
         29 . The bacterial cell according to  claim 27 , wherein part i) is physically linked to the DNA of at least one gene of interest, optionally with DNA restriction cleavage sites in between part i) and the at least one gene of interest. 
     
     
         30 . A method for transformation of a bacterial cell with a transcription control cassette according to  claim 16  by
 a) providing a transcription control cassette according to  claim 16 ; 
 b) transforming a host cell with the transcription control cassette; 
 c) selecting the host cell that have been stably transformed with the transcription control cassette by using the respective selection agents; and 
 d) isolating the transformed host cell.